Petri dish

Bacteria were grown overnight in LB medium, adjusted to an optical density (OD) at 600 nm of 0.01 in LB broth, and 200 µL were incubated in a 96 well plate in a TECAN (Infinite^® 200 PRO, Männedorf, Switzerland) plate reader at 37 °C and 6 mm shaking amplitude. Every 15 min the absorbance at 600 nm was automatically measured for 24 h. LB medium alone and E28 wild type served as controls. Data are presented as mean of triplicate determinations.
Media used to assay swimming motility of E. coli contained 2.5% Miller’s LB Broth Base™ powder and 0.3% (w/v) agar bacteriological No. 1 (Thermo Fisher Scientific, Waltham, MA, USA). After solidification of 9 mL/petri dish (55 mm, Greiner Bio-One, Kremsmünster, Austria), 1 µL of the corresponding overnight culture was placed in the middle of one plate. Plates were incubated at 37 °C for 18 h and the diameter of colonies was measured.

Husbandry and experimental procedures for the collection of zebrafish embryos (OBI/WIK strains) were performed at UFZ Helmholtz Centre for Environmental Research (Leipzig, Germany) according to the standard protocol as described^{43 (link),44 (link),61 (link)}. Briefly, fertilized and normally developed embryos were sorted and selected using a dissecting microscope, rinsed with egg water (ISO water) several times, and kept in a Petri dish (100 mm × 20 mm; Greiner Bio-One) (N ≤ 100 embryos). ISO water for the standard fish embryo test was prepared as described earlier (ISO 7346-3 (1996) [80 mM CaCl₂·2H₂O, 20 mM MgSO₄·7H₂O, 31 mM NaHCO₃, 3.1 mM KCl), pH 7.5]. Embryos were kept in an incubator (Binder, Gmbh, Germany) (14 h light: 10 h dark, 28 °C), and examined daily. All procedures were in accordance with the German animal protection standards approved by the Government of Saxony, Landesdirektion Leipzig, Germany (Aktenzeichen 75-9185.64), and guidelines of the European Union, Directive 2010/63/EU which permits the use of zebrafish embryos up to 120 hpf. All reporting of studies involving use of zebrafish embryos follow the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines^{71 (link)}.

Cells were seeded at 10⁴ and 5 × 10⁴ cells/mL density on LOC platform in medium dedicated to the cell line (Table 2). LOC was placed in the Petri dish (Greiner Bio One, Kremsmünster, Austria) and transferred to the incubator (NU-5510 E, NuAire; conditions 37 °C, 5% CO₂). Cells were observed after 24, 72, and 120 h under light microscope (Olympus IX81, Tokyo, Japan). In the subsequent experiment cell lines that were able to grow on the LOC were slowly adapted to CO₂-independent medium (cat. no. 18045-088; Life Technologies, Renfrew, UK), supplemented with 10% (v/v) FBS (GE Healthcare HyClone, Logan, UT, USA), 2 mM L-glutamine and antibiotics—100 µg/mL streptomycin and 100 U/mL penicillin by increasing in every passage (cells were passaged twice a week) the content of the CO₂-independent medium in relation to the standard medium. When cells were able to proliferate in 100% CO₂-independent medium, they were seeded at 10⁴ and 5 × 10⁴ on a LOC platform. Cells were observed after 24, 72 and 120 h under light microscope.

To estimate the number of mesenchymal clonogenic cells, a colony-forming unit fibroblast (CFU-F) assay was performed. Briefly 5000 cells were plated in a Petri dish (100 mm diameter, Greiner) with culture medium for 10 days. After May-Grünwald/Giemsa staining, colonies were defined as more than 50 fibroblastic cells and scored using an inverted microscope.

For biofilm growth on surface materials, 3 mL of the working culture was pipetted into a petri-dish (35 × 10 mm; Greiner Bio-One, Kremsmünster, Austria) and biofilm formation was analyzed up to 192 h, with orbital shaking (60 rpm) at 30 °C for M. lacticum or without shaking at 37 °C for S. capitis. Non-adherent bacterial cells were removed at 24, 48, 72, and 144 h by gently rinsing twice with 3 mL of phosphate-buffered saline (PBS, pH 7.4, composition: 0.14 M of NaCl, 2.7 mM of KCl, 10 mM of phosphate; Carl Roth, Karlsruhe, Germany). The biofilms were incubated accordingly.

FEP capillaries consisting of HeLa (stably expressing H2B‐mCherry, or H2B‐mCherry and LifeAct‐GFP) aggregates that had undergone a burst‐like cell dispersion within 8 h of sample preparation (Experimental Section), were continued to be stored at 37 °C in a humidified atmosphere with 5% CO₂ from days 1–4 (N = 10, for each day). After each day, the capillaries were removed and samples were pushed out carefully into a Petri dish (6015 mm, Greiner Bio‐one), after which 100 µL of culture medium was added on top of each of the aggregates (still in LCC) to prevent drying out. From here on, the samples were protected from light, and DRAQ‐7 (Thermofisher) was administered at 1:100 ratio to every sample droplet, and the Petri dish was stored at room temperature for 10 min. Spinning disk confocal images of the nuclei with lasers 561 nm and 647 nm (DRAQ‐7) were acquired with the Slidebook 6 software (3i) using an inverted microscope (Nikon Eclipse Ti‐E) equipped with a CSU‐W1 spinning disk head (Yokogawa) and a scientific CMOS camera (Prime BSI, Photometrics).
Nuclei were detected using Laplacian of Gaussian detection algorithm from Trackmate.^[47] The number of dead cells was subtracted from the total number of cells detected, to retrieve the % live cells.